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Cone-rod dystrophies (CRD) are forms of inherited retinal dystrophy which characteristically lead to early impairment of vision. An initial loss of colour vision (cone mediated functions) and of visual acuity, usually from the first or second decade of life, is followed by night blindness (largely rod mediated) and loss of peripheral visual fields.1 CRD patients suffer from severe photophobia and show reduced ERG responses. In later life, vision may be reduced to a bare perception of light. CRD is a milder condition compared to Leber congenital amaurosis (LCA) which is the most severe form of all the inherited retinal dystrophies and is diagnosed as bilateral congenital blindness, with a diminished or absent electroretinogram (ERG). Cone-rod dystrophy loci have been mapped to chromosomes 17q,2 19q,3 18q,4 17p13,5,6 6q,7 1q12,8 and 8p11.9 Mutations in the peripherin/RDS,10CRX,11,12 and RetGC-I13,14 genes have been shown to cause autosomal dominant CRD. Mutations in the ATP binding cassette transporter rim protein (ABCR) gene have been shown to be associated with autosomal recessive CRD.15 Mutations in the CNGA3 gene encoding the α-subunit of the cone photoreceptor cGMP gated channel have also been reported to cause cone photoreceptor disorders.16
The RPGRIP1 protein (retinitis pigmentosa GTPase regulator interacting protein 1, MIM 605446) is encoded by the gene located on chromosome 14q11. It consists of 24 exons and the predicted size of its protein product is 1259 amino acids. It is expressed specifically in the rod and cone photoreceptors and is a structural component of the ciliary axoneme. One of its functions is to anchor the RPGR protein within the photoreceptor connecting cilium.17 Recently, in an in vivo investigation of RPGRIP1 function and its physical interaction, it has been shown that RPGRIP1 is essential for RPGR function and is also required for normal disk morphogenesis.18 Mutations in RPGRIP1 have been reported to be a cause of LCA.19,20 Here we report the first observation of the involvement of RPGRIP1 gene mutations as a cause of CRD in four Pakistani families.
SUBJECTS AND METHODS
We studied 20 members of a two generation and 19 members of a three generation consanguineous Pakistani families, 1CRD and 4CRD, respectively. The 1CRD family consists of eight affected and 12 unaffected subjects and the 4CRD family consists of eight affected and 11 unaffected subjects (fig 1A, B). One of the authors (A Aziz) clinically examined all the patients and their unaffected family members. The deterioration in central vision and colour blindness was from an early age in all the patients and there was a rapid loss of vision between the ages of 14 and 16 years (visual acuity 1/60, 0.01). Patients had also had severe photophobia since their childhood. Fundoscopy showed a variable degree of fundus granularity and macular degeneration. The affected subject IV.7 (aged 14 years) has a characteristic macular bull’s eye lesion in both her eyes. Full field flash ERG was used to measure functions of both cones and rods. Both photopic and scotopic full field ERG amplitudes were reduced, showing involvement of both photoreceptor systems. However, among the young patients, cone response was reduced more than that of their rod response. Based on family history and clinical diagnosis, the disease was classified as autosomal recessive cone-rod dystrophy (arCRD).
For genetic analysis peripheral blood samples were collected with informed consent from all members of the two families and from 100 ethnically matched control subjects.
Genomic DNA was extracted from whole blood using the Nucleon II extraction kit (Scotlab Bioscience). DNA was amplified using primers (Research Genetics) for polymorphic microsatellite markers specific for known loci/genes associated with various retinal degenerations according to the conditions described previously.8
Exon specific intronic primers were designed from the genomic sequence of the RPGRIP1 gene (NM 020366). PCR was performed in a 50 μl reaction volume using 1 μg of genomic DNA. The resulting product was allowed to cool slowly to room temperature to maximise the formation of heteroduplexes. Heteroduplex analysis was performed using an automated DHPLC instrumentation (WAVE DNA fragment analysis system, Transgenomic, Crewe, UK). Sample preparation for heteroduplex analysis was carried out by denaturing and reannealing of unpurified PCR products of the carriers (heterozygotes). The temperature conditions required for the successful resolution of heteroduplexes were obtained from the website (http://insertion.stanford.edu/melt.html).
On identification of heteroduplex peaks in the carriers, all family members were sequenced in the forward and reverse directions using a commercially available kit (Big Dye, ABI) and the products were analysed on an ABI Prism 377 automated DNA sequencer. Subsequently, six small families with CRD (5, 6, 7, 8, 9, and 10CRDs) and two with LCA were also included for mutation screening of the RPGRIP1 gene. To exclude the possibility that the mutations are polymorphisms, 100 ethnically matched control samples were also screened for heteroduplexes. The unpurified PCR products of the control samples and the homozygous wild type reference DNA sample were mixed in an equimolar ratio. The mixture was then subjected to a three minute, 95°C denaturing step followed by gradual reannealing from 95–65°C over 30 minutes. The heteroduplex mismatches were detected using the WAVE DNA fragment analysis system.21
RPGRIP1 gene mutations have previously been reported to cause Leber congenital amaurosis.
Here we report two novel mutations in the RPGRIP1 gene that cause cone-rod dystrophy in four Pakistani families.
A homozygous G to T point mutation was identified in exon 16 at nucleotide 2480 in all the affected members of family 1CRD.
In the affected members of another CRD family (4CRD), a G to T substitution was found in exon 13 at nucleotide 1639.
The same G 1639 T mutation was found in two more small families, 5CRD and 10CRD.
Exclusion studies on both the large families (1CRD and 4CRD) showed linkage with the microsatellite marker D14S1023 (Zmax = 5.17 and 4.21 for the two families respectively at θ = 0.00) at chromosome 14q11, a locus for the RPGRIP1 gene (fig 1A, B). Mutation screening of the candidate gene, RPGRIP1, was carried out to identify the disease associated mutations.
Initially, DHPLC analysis was performed with samples from unaffected carriers of the families. Heteroduplex mismatches were recognised by the appearance of more than one peak in the elution profile. The presence of heteroduplex peaks in the unaffected carriers was convincing enough to do sequencing to identify the exact mutational change.
Sequence analysis of the RPGRIP1 gene for family 1CRD showed a homozygous G to T point mutation in exon 16 in all the affected subjects (fig 2A). This substitution at nucleotide 2480 changes codon 827 from CGC (Arg) to CTC (Leu). This change in exon 16 was not found in any other Pakistani family studied here, the 100 control samples, nor any of the unaffected subjects of family 1CRD.
In the affected members of family 4CRD, a G to T substitution was found in exon 13 at nucleotide 1639 (fig 2B). This point mutation changes codon 547 from GCT (Ala) to TCT (Ser). The same mutation was found in two other small families, 5CRD and 10CRD (fig 2C). No disease associated mutation was observed in the RPGRIP1 gene sequence for the remaining CRD and LCA families examined.
In addition, three polymorphisms were also identified in the RPGRIP1 gene, which include a CTC to CTT (Leu427Leu) polymorphism in exon 16, a G to A sequence change in intron 9 and a deletion (9 base pair) in the intronic region of exon 13, 32 bp downstream from exon 13.
Homozygous mutations in the RPGRIP1 gene have been reported in a panel of unrelated patients with Leber congenital amaurosis (LCA).20 In most of these cases, the mutations result in a premature termination codon. To date RPGRIP1 is the only gene that has not been associated with any other retinal disease phenotypes except LCA. LCA represents the severe end of a spectrum of inherited retinal dystrophies while cone-rod dystrophy is a milder condition. It has been suggested that mutations that cause residual RPGRIP1 activity may lead to phenotypes such as RP or CRD which are less severe compared to LCA.22 Mapping of two Pakistani families with cone-rod dystrophy to the RPGRIP1 locus supports this hypothesis. The identification of two novel disease associated mutations also indicates allelic heterogeneity of the RPGRIP1 gene. Both novel mutations are in exons encoding domains of RPGRIP1 that are reported to be involved in interaction with the RPGR gene product.20 Prosite (http://us.expasy.org/tools/scanprosite/) scan predicted the occurrence of an additional, more efficient, casein kinase II phosphorylation site in the Ala547Ser mutated protein, the significance of which is unknown. The second mutation (Arg827Leu) occurs in the major calcium dependent membrane binding module, the CK2 domain of the RPGRIP1 protein. However, the prediction does not indicate any change in the 3D structure of the domain. Functional analysis of this protein would be required to demonstrate the role of these mutations in retinal dystrophies.
The authors thank the family members for taking part in this study. We also thank Dr Q Ayub for his valuable help. This work was supported by Wellcome Trust grant number 063406/Z/2000/Z to SQM.
The first two authors contributed equally to this work.